a simplified method for extraction of ergosterol from soil

4
DIVISION S-3-SOIL BIOLOGY & BIOCHEMISTRY A Simplified Method for Extraction of Ergosterol from Soil N. S. Hash,* P. D. Stahl, T. B. Parkin, and D. L. Karlen ABSTRACT Ergosterol is a sterol common to many fungi and may be useful for estimating fungal biomass in soil. Our objective was to compare three different methods for extracting ergosterol from soil. These included: (i) a published method that involves extraction with metha- nol, saponification with KOH, and separation with hexane before analysis using high-pressure liquid chromatography (HPLC); (ii) a recently published supercritical fluid extraction (SFE) method; and (iii) a simplified extraction method developed in our laboratory that is similar to method (i) but requires 80 to 90% less reagents. Similar quantities of ergosterol were extracted from both prairie and cropland soil with method (i) and with simplified method (iii). In contrast, the SFE method (ii) did not recover naturally occurring ergosterol from soil. Extraction efficiencies of ergosterol standards added to soil were between 75 and 88% for the three procedures, with coefficients of variation of <15% for all methods. Our simplified method substantially reduced cost, extraction time, and chemical waste per sample. Results of this study indicate that the simplified method was the most efficient technique for extraction of ergosterol from soil. F UNGI play an important role in ecosystem function (Christensen, 1989) and contribute significantly to soil quality (Eash et al., 1994). They are a major compo- nent of the soil microbial community and represent a dynamic pool of nutrients in the form of living and dead microorganisms (Doran and Linn, 1994). Fungi are the primary decomposers of plant residue, mineralizing and immobili/ing important nutrients that support successive crops following lysis, death, and decomposition (Baath and Soderstrom, 1979; Paul and Clark, 1989). Fungi serve as a food source for soil fauna, are an important mediator in soil aggregate formation (Eash, 1993; Jas- trow and Miller, 1991; Molope, 1987; Tisdall and Oades, 1982), and cause disease in plants. Until accurate and reliable methods for evaluating fungal biomass in soil are determined, the role of fungi as "conservers" and "cyclers" of nutrients cannot be accurately quantified (Franklandetal., 1990; Newell, 1992; Parkinson, 1994). Current methods for assessing fungal presence in soil include direct microscopy, ergosterol extraction, immu- noassays, enzyme activities, and selective inhibition assays (Newell, 1992). The usefulness of these methods, however, is disputed due to problems intrinsic to each. Direct microscopy is the most common technique to N.S. Eash, Plant and Soil Science Dep., Univ. of Tennessee, Knoxville, TN 37901; P.O. Stahl, T.B. Parkin, and D.L. Karlen, USDA-ARS, National Soil Tilth Lab., 2150 Pammel Drive, Ames, IA 50011. Mention of trademark, proprietary product, or vendor does not constitute a guarantee or warranty of this product by the USDA and does not imply its approval to the exclusion of other products or vendors that may also be suitable. Received 24 Oct. 1994. *Corresponding author ([email protected]). Published in Soil Sci. Soc. Am. J. 60:468-471 (1996). estimate soil fungal biomass, but results are often influ- enced by observer subjectivity (Stahl et al., 1995; New- ell, 1992). Estimation of fungal biomass from soil ergo- sterol concentration is gaining in popularity but requires the use of conversion factors that are not yet well estab- lished. Results of previous studies (Stahl et al., 1995; West et al., 1987) suggest that using direct counts in combination with ergosterol measurements can increase the reliability and accuracy of estimating fungal biomass. Use of both methods can also aid in the comparison of estimates from independent laboratories. Ergosterol is an indicator of live fungal biomass (West et al., 1987) because it is quickly degraded. Davis and LaMar (1992) found >95% reduction in ergosterol con- tent within 2 wk following fumigation and death of the fungal cells. Ergosterol correlates with fungal surface area (West et al., 1987) and hyphal length (Matcham et al., 1985), and is more sensitive than chitin or extracellu- lar laccase assays (Matcham et al., 1985; Seitz et al., 1979). Given that ergosterol is the most frequently en- countered fungal sterol (Davis and LaMar, 1992; Grant and West, 1986; Mercer, 1984; Peacock and Goosey, 1989; Pierce et al., 1979; Weete, 1973), ergosterol may be a good indicator of fungal biomass in soil. The most commonly used method for extracting ergo- sterol from soil (Grant and West, 1986) is resource intensive because it requires large volumes of solvent and a time-consuming reflux step. A recently published method that potentially overcomes these limitations is SFE (Young and Games, 1993). This method accom- plishes extraction using liquid CC*2 as the solvent; thus, no hazardous wastes are generated. However, the SFE method was developed for grain samples and has not been tested on soil. As an alternative to the Grant and West method, we developed a simplified procedure for ergosterol extraction from soil. The objective of this study was to compare these three methods for extraction of ergosterol from soil. To accomplish this goal, we performed two experiments, one in which the three meth- ods were compared with regard to extraction of naturally occurring ergosterol from each of two soils, and another where we evaluated the efficiency of the methods by measuring recovery of ergosterol standards added to soil. MATERIALS AND METHODS Three methods for ergosterol extraction from soil were evaluated with respect to extraction efficiency and variability. The methods compared were: (i) the technique of Grant and West (1986), (ii) the SFE as described by Young and Games Abbreviations: HPLC, high pressure liquid chromatography; SFE, super- critical fluid extraction; CV, coefficient of variation; ANOVA, analysis of variance. 468

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DIVISION S-3-SOIL BIOLOGY & BIOCHEMISTRY

A Simplified Method for Extraction of Ergosterol from SoilN. S. Hash,* P. D. Stahl, T. B. Parkin, and D. L. Karlen

ABSTRACTErgosterol is a sterol common to many fungi and may be useful

for estimating fungal biomass in soil. Our objective was to comparethree different methods for extracting ergosterol from soil. Theseincluded: (i) a published method that involves extraction with metha-nol, saponification with KOH, and separation with hexane beforeanalysis using high-pressure liquid chromatography (HPLC); (ii) arecently published supercritical fluid extraction (SFE) method; and(iii) a simplified extraction method developed in our laboratory thatis similar to method (i) but requires 80 to 90% less reagents. Similarquantities of ergosterol were extracted from both prairie and croplandsoil with method (i) and with simplified method (iii). In contrast, theSFE method (ii) did not recover naturally occurring ergosterol fromsoil. Extraction efficiencies of ergosterol standards added to soil werebetween 75 and 88% for the three procedures, with coefficients ofvariation of <15% for all methods. Our simplified method substantiallyreduced cost, extraction time, and chemical waste per sample. Resultsof this study indicate that the simplified method was the most efficienttechnique for extraction of ergosterol from soil.

FUNGI play an important role in ecosystem function(Christensen, 1989) and contribute significantly to

soil quality (Eash et al., 1994). They are a major compo-nent of the soil microbial community and represent adynamic pool of nutrients in the form of living and deadmicroorganisms (Doran and Linn, 1994). Fungi are theprimary decomposers of plant residue, mineralizing andimmobili/ing important nutrients that support successivecrops following lysis, death, and decomposition (Baathand Soderstrom, 1979; Paul and Clark, 1989). Fungiserve as a food source for soil fauna, are an importantmediator in soil aggregate formation (Eash, 1993; Jas-trow and Miller, 1991; Molope, 1987; Tisdall and Oades,1982), and cause disease in plants. Until accurate andreliable methods for evaluating fungal biomass in soilare determined, the role of fungi as "conservers" and"cyclers" of nutrients cannot be accurately quantified(Franklandetal., 1990; Newell, 1992; Parkinson, 1994).

Current methods for assessing fungal presence in soilinclude direct microscopy, ergosterol extraction, immu-noassays, enzyme activities, and selective inhibitionassays (Newell, 1992). The usefulness of these methods,however, is disputed due to problems intrinsic to each.Direct microscopy is the most common technique to

N.S. Eash, Plant and Soil Science Dep., Univ. of Tennessee, Knoxville,TN 37901; P.O. Stahl, T.B. Parkin, and D.L. Karlen, USDA-ARS,National Soil Tilth Lab., 2150 Pammel Drive, Ames, IA 50011. Mentionof trademark, proprietary product, or vendor does not constitute a guaranteeor warranty of this product by the USDA and does not imply its approvalto the exclusion of other products or vendors that may also be suitable.Received 24 Oct. 1994. *Corresponding author ([email protected]).

Published in Soil Sci. Soc. Am. J. 60:468-471 (1996).

estimate soil fungal biomass, but results are often influ-enced by observer subjectivity (Stahl et al., 1995; New-ell, 1992). Estimation of fungal biomass from soil ergo-sterol concentration is gaining in popularity but requiresthe use of conversion factors that are not yet well estab-lished. Results of previous studies (Stahl et al., 1995;West et al., 1987) suggest that using direct counts incombination with ergosterol measurements can increasethe reliability and accuracy of estimating fungal biomass.Use of both methods can also aid in the comparison ofestimates from independent laboratories.

Ergosterol is an indicator of live fungal biomass (Westet al., 1987) because it is quickly degraded. Davis andLaMar (1992) found >95% reduction in ergosterol con-tent within 2 wk following fumigation and death of thefungal cells. Ergosterol correlates with fungal surfacearea (West et al., 1987) and hyphal length (Matcham etal., 1985), and is more sensitive than chitin or extracellu-lar laccase assays (Matcham et al., 1985; Seitz et al.,1979). Given that ergosterol is the most frequently en-countered fungal sterol (Davis and LaMar, 1992; Grantand West, 1986; Mercer, 1984; Peacock and Goosey,1989; Pierce et al., 1979; Weete, 1973), ergosterol maybe a good indicator of fungal biomass in soil.

The most commonly used method for extracting ergo-sterol from soil (Grant and West, 1986) is resourceintensive because it requires large volumes of solventand a time-consuming reflux step. A recently publishedmethod that potentially overcomes these limitations isSFE (Young and Games, 1993). This method accom-plishes extraction using liquid CC*2 as the solvent; thus,no hazardous wastes are generated. However, the SFEmethod was developed for grain samples and has notbeen tested on soil. As an alternative to the Grant andWest method, we developed a simplified procedure forergosterol extraction from soil. The objective of thisstudy was to compare these three methods for extractionof ergosterol from soil. To accomplish this goal, weperformed two experiments, one in which the three meth-ods were compared with regard to extraction of naturallyoccurring ergosterol from each of two soils, and anotherwhere we evaluated the efficiency of the methods bymeasuring recovery of ergosterol standards added to soil.

MATERIALS AND METHODSThree methods for ergosterol extraction from soil were

evaluated with respect to extraction efficiency and variability.The methods compared were: (i) the technique of Grant andWest (1986), (ii) the SFE as described by Young and Games

Abbreviations: HPLC, high pressure liquid chromatography; SFE, super-critical fluid extraction; CV, coefficient of variation; ANOVA, analysisof variance.

468

EASH ET AL.: EXTRACTING ERGOSTEROL FROM SOIL 469

(1993), and (iij) a simplified method developed in our labora-tory. In one experiment, direct comparison of the three methodswas conducted by comparing amounts of naturally occurringergosterol extracted from soil. In this experiment, a nativeprairie soil and a cultivated soil were used. In a second experi-ment, extraction efficiency of the three methods was evaluatedby measuring recovery of ergosterol standards added to soil.

Comparative Extraction ExperimentsThe three methods were compared for extraction of naturally

occurring ergosterol from two soil samples. Both sampleswere Kossuth silty clay loam (fine-loamy, mixed, mesic TypicHaplaquolls), one collected from an undisturbed prairie site(Doolittle Prairie Preserve in Story Country, Iowa) and thesecond from a cultivated field under a corn-soybean rotationlocated 25 m from the prairie site. Three field-moist replicateswere extracted from both samples using the three extractionprocedures described below.

Spike-Recovery ExperimentsErgosterol recovery from soil was evaluated by spike-

recovery experiments using three slightly different procedures.For the Grant and West method, five replicate 25-g field-moistsamples of Sable silty clay loam soil (fine-silty, mixed, mesicTypic Haplaquolls) were spiked with 100 ng of ergosterol(Sigma Chemical, St. Louis, MO). Ergosterol dissolved inmethanol was added to moist soil immediately before extrac-tion. Samples were extracted according to the published methodof Grant and West (1986) outlined below. Evaluation of theSFE method was performed by adding 50 u,g of ergosterol to5 g of field-moist Kossuth silty clay loam in the SFE thimble(three replicates). Samples were then extracted for ergosterolaccording to the published method of Young and Games (1993)as described below. Evaluation of our simplified extractionprocedure was conducted by spiking four replicate 5-g samplesof field-moist Saybrook silt loam (fine-silty, mixed, mesicTypic Argiudolls) with 25 |ig of ergosterol and extractedfollowing the procedure outlined below.

Grant and West MethodErgosterol extraction and measurement followed the proce-

dure of Grant and West (1986) with minor modifications.Field-moist soil (25 g) was passed through a 4-mm sieveinto 125-mL round high-density polyethylene Nalgene bottles.Eighty milliliters of HPLC-grade cold (0°C) methanol wereadded and samples were sonicated (6 min at medium power,10% duty cycle, extended probe, Sonics and Materials, Dan-bury, CT). The soil-methanol mixture was filtered throughWhatman no. 41 filter and washed with five 10-mL aliquotsof methanol into 250-mL Erlenmeyer flasks. On a magneticstirrer, 12 g of KOH and 24 mL of ethanol (95%) were added.Samples were saponified under reflux (approximately 60°C)for 30 min, cooled, and 30 mL of deionized water was added.Ergosterol was partitioned from the sample with three 80-mLaliquots of hexane in a 500-mL separatory funnel. The hexaneextract was concentrated on a rotary evaporator for 5 to 10min under N2 gas .until 5 to 20 mL remained. The samplewas then transferred and dried in an automated evaporationworkstation (TurboVap, Zymark Corp., Hopkinton, MA) un-der N2 gas.

The sample was dissolved in 10 mL of methanol, homoge-nized with a vortex mixer, and reduced to 1- to 2-mL volumein the evaporator. The methanol extract was drawn from theevaporator tubes with a 5-mL syringe (tared) and expelled

through a 0.20-um syringe filter into the HPLC vials. Thevolume of the methanol extract was determined on a weightbasis. Samples were stored in the freezer (0°C) until analysis.Following this procedure, 12 samples was the maximum num-ber one person could process per day.

Supercritical Fluid Extraction MethodThe method recently published by Young and Games (1993)

was used. Soil (5 g) was placed into a Hewlett-Packard 7680ASFE module (Hewlett-Packard, Avondale, PA). Ergosterolwas extracted using a 40°C extraction chamber with supercriti-cal CO2 at a density of 0.90 g/mL (28 MPa) for 10 min at aflow rate of 3 mL/min (equivalent to 4.3 thimble volumes).Analytes from the extraction chamber were trapped on a C-18column at 40°C. The trap was then heated to 50°C and rinsedwith 1.8 and 1.9 mL of methanol on two successive rinses ata flow rate of 1.01 mL/min.

Simplified MethodThe simplified ergosterol extraction method was a modifica-

tion of the Grant and West method. Soil (5 g), 15 mL cold(0°C) methanol, and 5 mL of solution (40 g/L KOH in 95%ethanol) were placed into 16 by 125 mm centrifuge tubes,homogenized on a vortex mixer for a few seconds, and soni-cated (1 min at medium power, 10% duty cycle, extendedprobe, Sonics and Materials, Danbury, CT). The extractionmixture was transferred to 35-mL pressure reaction tubes(Alltech, Deerfield, IL) and placed in an 85°C water bath for30 min, removing after 15 min to vortex mix contents. Tubeswere cooled and deionized water (5 mL) was added. Theextraction mixture was filtered through Whatman no. 41 paper,which was rinsed with 5 mL of methanol. The filtrate wastransferred to 250-mL separatory funnels and extracted withpentane three times (3 X 10 mL). The pentane layer (top)was removed and placed into evaporation tubes. The tubeswere placed into the evaporator (60°C) and dried under N2gas. The sample was dissolved in 5 mL of methanol, homoge-nized with a vortex mixer, and reduced to 1 to 1.5 mL. Themethanol extract was removed from the tubes with a 5-mLsyringe (tared) and expelled through a 0.20-u,m syringe filterinto amber HPLC vials. The methanol extract volume wasdetermined on a weight basis. More than 24 samples couldbe extracted per day by one operator with limited numbers(six) of pressure reaction tubes. With adequate glassware, thenumber of samples per day could be increased.

Ergosterol QuantificationExtracted ergosterol was analyzed on a LiChrospher 100

RP-18 column (Hewlett-Packard, Avondale, PA) at40°C col-umn temperature using a methanol/water (95:5 v/v) mobilephase, a variable flow rate of 0.5 to 2 mL/min and UV detectionat 282 nm with a Hewlett Packard 1090A HPLC equippedwith a diode array detector and a 250-p.L injection loop.Ergosterol retention time was approximately 7 min. Ergosterolstandards (Sigma Chemical, St. Louis, MO) were used todetermine the lower quantitation limit (0.005 ng/nL injected).

Statistical AnalysesResults were analyzed using a one-way repeated measures

ANOVA test for significance. Treatment differences were eval-uated using a Bonferroni r-test (P < 0.05).

470 SOIL SCI. SOC. AM. J., VOL. 60, MARCH-APRIL 1996

Table 1. Soil ergosterol contents following various extraction pro-cedures.

Method Prairie soil Cornfield soil

mg kg-'PrimarySimplifiedSupercritical fluid extract

4.47at5.63a (4.3)

ND§

3.01a (11.8)3.17a (8.4)

ND

t Means with the same letter are not significantly different (P < 0.05).Comparisons are between methods using the same soil type.| Data are means with coefficients of variation in parentheses.§ Ergosterol was not detected in the sample.

RESULTS AND DISCUSSIONExtraction of naturally occurring ergosterol from two

soils indicated that the three methods performed differ-ently (Table 1). For each of the soils, the method ofGrant and West and our simplified method extractedsimilar amounts of ergosterol. Soil ergosterol concen-trations determined by these two methods were 4.5and 5.6 mg/kg for the prairie soil and approximately 3mg/kg for the cultivated soil, with no significant differ-ences between methods.

No ergosterol was extracted from either soil using theSFE method. This method was originally developed forergosterol determination in grain samples, and not foruse with soil. The reasons for failure of the method toextract naturally occurring ergosterol were not apparentbut similar to the results of Young and Games (1993).

A key component of our comparison was determinationof efficiencies of three different ergosterol extractionmethods. Spike-recovery experiments indicate that allthe methods had similar extraction efficiencies (Table2). Recovery of added ergosterol ranged from 69 to 88%for the three methods.

Other studies in the literature have evaluated extractionefficiency of ergosterol from only soil methanol extractsinstead of adding ergosterol spikes to the soil sampleprior to beginning the extraction process. Grant and West(1986) had an 8% loss of ergosterol added to methanolextracts while Newell et al. (1988) reported recoveriesin the range of 95 to 107% from methanol extracts ofplant materials. While evaluating this part of the extrac-tion process is important, addition of ergosterol to soilprior to chemical extraction allows evaluation of thecomplete chemical extraction and analysis process. Inanother study, Davis and LaMar (1992) found a 76%recovery of ergosterol added to the soil prior to extrac-tion, results that are very similar to those found inthis study. Whereas determining recovery efficiencies of

Table 2. Extraction efficiency of ergosterol spikes from soil orsand using various methods.

Method nt Recovery Soil CV

PrimarySimplifiedSFE§SFESFE

54333

79.3 (4.63»87.6 (6.86)69.4 (10.05)81.0 (3.52)95.4 (7.54)

SableSaybrookKossuth prairieKossuth corn fieldsand

5.87.8

14.44.47.9

t Number of samples.I Data are means with standard deviations in parentheses.§ SFE = supercritical fluid extraction method.

added ergosterol in spike-recovery evaluations is a usefulway to compare methods, it is also necessary to compareextraction of naturally occurring ergosterol from fungaltissue in soil (Davis and LaMar, 1992).

Ergosterol can be present in either bound or free form(Seitz et al., 1977). In a study evaluating fungal growthin grain, Seitz et al. (1977) suggested that free ergosterolcan be extracted without saponification while saponifica-tion is necessary to liberate esterified ergosterol deriva-tives. The SFE method did successfully extract ergosterolspikes in the spike-recovery experiment. While the SFEmethod appears to work well with grain samples (Youngand Games, 1993) or extracting ergosterol spikes fromsoil, the interaction of bound fungal ergosterol with thesoil matrix may require a harsher extraction procedure.

Close evaluation of ergosterol extraction data suggestthat there may be a trend of higher ergosterol extractionusing our simplified method. Unpublished results fromour lab indicated that the saponification step is criticalin the extraction process. In our simplified method, KOHis added at the onset of the extraction process insteadof following the sonication step as outlined by Grant andWest (1986). The increased exposure time of the sampleto the KOH may increase extraction efficiency; however,additional data are needed to confirm this hypothesis.

Pentane was used instead of hexane in our simplifiedmethod because it quickly volatilizes in the evaporator.Earlier unpublished experiments comparing hexane andpentane extraction efficiencies indicated that either sol-vent extracted similar amounts of ergosterol.

Evaluation of variability is another important criterionfor assessment of extraction techniques. Variability asso-ciated with all three methods was similar. In the spike-recovery experiments, CVs ranged from 5.8 to 14.4%while CVs for extraction of naturally occurring ergosterolranged from 4.3 to 14.6%.

CONCLUSIONSIn selecting an optimum procedure, several factors

must be considered. Extraction efficiency of added ergo-sterol between each of the three methods was comparable,with similar variability associated with all three methods.However, our results indicate that the SFE method isnot suitable for extraction of natural ergosterol fromsoil. The simplified ergosterol assay we developed hasadvantages over the method of Grant and West (1986).Approximately 80 to 90% less reagents are used, re-sulting in reduced cost and less chemical waste. Theextraction step of our method requires only 15 to 20 mLof hexane or pentane as opposed to 240 mL of hexaneprescribed by the Grant and West (1986) technique. Inaddition, our simplified technique has eliminated severaltime-consuming steps associated with that method. Dueto smaller sample size, sonication is done in centrifugetubes for 1 min instead of 6 min. Individual refluxingof each sample is eliminated because a rack of samplesis heated simultaneously in an 85 °C water bath for 30min, resulting in a substantial decrease in the amountof time and equipment needed.

HASH ET AL.: EXTRACTING ERGOSTEROL FROM SOIL 471

ACKNOWLEDGMENTSThe authors are extremely grateful to Richard Pfeiffer and

Tim Watts for their expertise and assistance.